Dual delivery nanoscale device for miR-345 and gemcitabine co-delivery to treat pancreatic cancer

Abstract

A polymeric dual delivery nanoscale device (DDND) was designed for combined delivery of microRNA (miR-345) and gemcitabine (GEM) to treat pancreatic cancer (PC). This temperature and pH-responsive pentablock copolymer system was able to restore miR-345, making xenograft tumors more susceptible to GEM, the standard therapy for PC. Restoration using DDND treatment results in sonic hedgehog signaling down regulation, which decreases desmoplasia, thereby resulting in improved GEM perfusion to the tumor and better therapeutic outcomes. The release of miR-345 and GEM could be tuned by using the DDND in the form of micelles or in the form of thermoreversible gels, based on polymer concentration. The DDNDs enabled miR-345 stability and sustained co-release of miR-345 and GEM, thereby facilitating dose-sparing use of GEM. Further, enhanced in vitro cellular uptake due to amphiphilic character, and endosomal escape because of the cationic end blocks led to efficient transfection with DDNDs. The combined DDND treatment enabled efficient reduction in cell viability of Capan-1 and CD18/HPAF cells in vitro compared with either GEM or miR-345 treatment alone. Mice carrying xenograft tumors treated with DDNDs carrying both miR-345 and GEM combination therapy displayed reduced tumor growth and less metastasis in distant organs compared to individual drug treatments. Immunohistochemical analysis of the xenograft tissues revealed significant down regulation of desmoplastic reaction, SHH, Gli-1, MUC4, and Ki67 compared to control groups.

Keywords: Gemcitabine; Nanoscale delivery; Pancreatic cancer; miR-345.

Figure 1.

Stability of DDND systems at different N/P ratios in media containing 10% serum and 0.25% RNase enzyme. Incubation time: 72h. Size and zeta potential stability of the DDND system with N/P ratio of 60 in 10% serum containing cell culture media.

Figure 2.

Release profiles of (A) Gemcitabine and (B) miR-345 from DDND micelles with N/P ratios of 20, 40 and 60. Release profiles of (C) Gemcitabine and (D) miR-345 from DDND gels with N/P ratios of 20, 40 and 60.

Figure 3.

Effect of DDND systems with N/P ratios of 60, 40 and 20 on (A) Capan-1 and (B) CD18/HPAF PC cell viability. Cell Density: 1×10⁴ cell/well. miRNA Dose: 10 pmol. Gemcitabine dose: 3 μg/well. Incubation time: 72h. The same letters represent statistically significant differences between cases (p<0.05).*, ** and *** represent the statistically significant differences of the DDND system with N/P ratios of 60 against all controls (p<0.05).

Figure 4.

Confocal images of DDND systems prepared at N/P ratio of 60 using dye attached PB copolymer in (A) Capan-1 and (B) CD18/HPAF cells. Incubation time: 24 h. Red: DDND stained by Alexaflour647, Green: lysosome stained by Lysotracker Green, Blue: nucleus stained by Hoechst dye.

Figure 5.

Transfection of DDNDs prepared at N/P ratio of 60 in (A) Capan-1 and (B) CD18/HPAF cells. Luciferase expressing gwiz plasmid complexed with Lipofectamine was used as positive control. Luciferase expressing gwiz plasmid alone or complexed with PB copolymers in the presence or absence of GEM/Pluronic F127 self-assembly were used as controls. * represents statistically significant differences (p<0.05) while same letters represent statistically insignificant difference (p>0.05).

Figure 6.

Therapeutic potential of nanoparticle carrying GEM + miR-345 mimics on orthotopically grown PC tumors in athymic nude mice. (A) Luciferase live animal IVIS imaging to monitor growth of tumors during the treatment period (B) Tumor weights at study endpoints.

Figure 7.

Effect of DDNDs loaded with miR-345 and GEM on tumorigenicity and metastasis (A) Changes in distant organs with metastatic lesions and pancreatic tumor tissue histology as a result of DDND mediated delivery of miR-345 and GEM. The first and second rows are the light microscopy images (100×) of H&E stained metastatic lesions from the control group. The third row shows H&E stained primary pancreatic tissue sections for each treatment group. (B) The number of animals showing incidence of metastasis at distant organs in the various treatment groups.

Figure 8.

Analysis of tumor samples for SHH, Glil, Trichrome, MUC4, and Ki-67 staining for each treatment groups.